346 CHEMISTRY & BIODIVERSITY – Vol (2008) Constituents of Asarum sieboldii with Inhibitory Activity on Lipopolysaccharide (LPS)-Induced NO Production in BV-2 Microglial Cells by Ah-Reum Han, Hye Jeoung Kim, Minkyu Shin, Moochang Hong, Yang Seok Kim, and Hyunsu Bae* BK21 Oriental Medical Science Center, College of Oriental Medicine, Kyung Hee University, Hoeki-dong, Dongdaemun-gu, Seoul 130-701, Korea (phone: ỵ 82-2-961-0323; fax: ỵ 82-2-967-2080; e-mail: hbae@khu.ac.kr) Bioassay-guided fractionation of the root extract of Asarum sieboldii led to the isolation of the four active compounds ()-sesamin (1), (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), kakuol (3), and 13,4,5-trimethoxytoluene3 ( ¼ 1,2,3-trimethoxy-5-methylbenzene; 4), in terms of inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production Compounds – showed potent inhibition of NO production, with IC50 values in the low nanomolar-to-micromolar range Also isolated were the known compounds methylkakuol (5), 13,5-dimethoxytoluene3, safrole, asaricin, methyleugenol, and ()-asarinin, which were found to be inactive in the above assay Among the ten known isolates, compounds 1, 2, and were found for the first time in this plant Introduction – The Asarum species (Aristolochiaceae) are herbal plants distributed through North America, Europe, and Asia [1] The roots of these species were used in traditional medicine as antitussive [2], anti-allergic [3], antihyperlipemic [4], expectorant [5] [6], anti-inflammatory [7] [8], anesthetic [9], and antifungal agents [10] [11] Previous phytochemical work on Asarum species resulted in the isolation of various types of essential oils [5] [12] [13], amides [14] [15], lignans [14] [16], flavonoids [17] [18], terpenoids [19], and alkaloids [20] As a part of the search for new therapeutic agents from medicinal plants against inflammation and neurodegenerative diseases, the spray-dried extracts of 270 herbal medicines in the PhytoLibraryTM kit were primarily tested for their inhibitory activities towards lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 microglial cells As a result, the roots of A sieboldii, which showed considerable inhibitory activity (70.4% inhibition at mg/ml), were selected for activity-guided fractionation NO is a simple, inorganic, gaseous free radical that is produced by the oxidation of l-arginine catalyzed by NO synthase (NOS), with a wide range of physiological and pathological actions [21] Among the NOS family, inducible NOS (iNOS), in particular, is involved in overproduction of NO, which is associated with oxidative stress and with pathophysiological responses, including circulatory shock, inflammation, and carcinogenesis [22] Also, it can be expressed in response to pro-inflammatory agents such as interleukin 1b (IL-1b), tumor necrosis factor a (TNF-a), and LPS in various cell types, including macrophages, endothelial cells, and smooth-muscle cells [23] In addition, overproduction of NO in the brain released by microglial cells contributes to neuronal damages accelerating various neurological disorders such as G 2008 Verlag Helvetica Chimica Acta AG, ZJrich CHEMISTRY & BIODIVERSITY – Vol (2008) 347 Parkinson and Alzheimer [24] Therefore, any treatment for the pathological symptoms related to NO is considered as a clinical approach either in preventing or in treating inflammatory and neurodegenerative disorders [24] [25] Herein, we describe the isolation, structural identification, and biological properties of ten known isolates from A sieboldii, including the biologically active constituents ()-sesamin (1) [14], (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2) [14], kakuol (3) [10], and 13,4,5-trimethoxytoluene3 ( ¼ 1,2,3-trimethoxy-5methylbenzene; 4) [26], as well as the inactive compounds methylkakuol (5) [27], 13,5dimethoxytoluene3 ( ¼ 1,3-dimethoxy-5-methylbenzene) [28], safrole [29], asaricin [30], methyleugenol [12], and ()-asarinin [14] Results and Discussion – Chemistry The hexane-soluble fraction of the MeOH extract of the roots of Asarum sieboldii Miquel showed inhibitory activity, with 68.9% inhibition at a concentration of mg/ml This fractions was, thus, subjected to detailed phytochemical analysis, which resulted in the isolation of the above ten compounds, which were structurally elucidated by physical and spectroscopic methods, as well as by comparison of their data with those published in the literature In addition, the configuration of ()-sesamin (1) and ()-asarinin were confirmed by comparison of their optical rotations with published values [14] Compounds 1, 2, and were found in this species for the first time, and compound has not been isolated before from the genus Asarum Biological Activity All compounds obtained in the present study were evaluated for their inhibitory activity on NO production in LPS-activated BV-2 microglial cells Among the isolates, compounds – were found to be significantly active ()-Sesamin (1) exhibited the most potent inhibition with an IC50 value of 6.26 10 10 m However, it was cytotoxic at the tested concentrations of 2.82 10 to 2.82 10 m in the MTT 1) assay Therefore, its iNOS inhibitory activity seems to be related to its concomitant cytotoxic effect There have been some other reports on the inhibitory 1) MTT ¼ 3-(4,5-Dimethyl-1,3-thiazol-2-yl)-2,5-diphenyltetrazolium bromide 348 CHEMISTRY & BIODIVERSITY – Vol (2008) activity of sesamin on LPS-induced NO production in BV-2 microglial cells [31] [32], so that compound was considered as a positive control in the present study Compounds 2, 3, and inhibited the LPS-induced NO production in a dosedependent manner, with IC50 values of 1.52 10 9, 3.6 10 7, and 1.4 10 m, respectively (Figure) These compounds did not show cytotoxicity at the test concentrations, indicating their true iNOS inhibitory activity Especially, compound 4, which exhibited the lowest IC50 value, inhibited significant LPS-induced NO production, i.e., 75.9% inhibition at the maximum test concentration of mg/ml (5.49 10 m) Figure Effects of compounds – on LPS-induced NO production in BV-2 microglial cells Inhibition of NO production was calculated relative to negative control, and values represent mean S.D of three experiments performed in triplicate Although compounds and have very similar structures, the only difference being the presence of a hydroxy (OH) vs a methoxy (MeO) function ortho to the acyl group, only showed inhibitory activity on LPS-induced NO production, compound being inactive Therefore, we can assume that the MeO group in 14,5-(methylenedioxy)propiophenones3 plays an important role in terms of inhibitory activity The remaining five known compounds, 13,5-dimethoxytoluene3, safrole, asaricin, methyleugenol, and ()-asarinin, showed negligible inhibitory activities, i.e., less than 50% inhibition at the maximum test concentration of mg/ml Hence, these compounds were regarded as inactive CHEMISTRY & BIODIVERSITY – Vol (2008) 349 In conclusion, compounds – showed significant activities in our assay Thus, further study on their mechanism of action can be encouraged to derive novel therapeutic agents against inflammation and neurodegenerative diseases The effect of sesamin (1) on LPS-induced NO production in BV-2 microglial cells has been reported previously Hou et al [31], and Jeng et al [32] reported that this compound decreased nitrite accumulation with 50% inhibition at 20 mm in BV-2 microglial cells, and inhibited LPS-induced cytokine production by suppression of p38 mitogen-activated protein kinase and nuclear factor-kB, respectively, although the configuration of this compound was not indicated (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10tetraenamide (2) was reported to inhibit NO production in LPS-stimulated RAW 264.7 macrophage cells, with an ID50 value of mg/ml [33] Therefore, this is the first report on the evaluation of inhibitory activity on NO production of all isolates, except for 1, in LPS-activated BV-2 microglial cells This work was supported by a grant (R13-2007-019-00000-0) from the Medical Science and Engineering Research Center program of the Ministry of Science and Technology (MOST) and the Korea Science and Engineering Foundation (KOSEF), and by a grant (15024) from the Creation of Geriatric Natural-MediCluser program of the Korean Government, Seoul City Experimental Part General Flash column chromatography (FC): silica gel 60 (70 – 230 mesh; Merck) TLC: precoated Kieselgel 60 F254 plates (0.25 mm; Merck); visualization under UV light (254 and 365 nm) and by spraying with 10% (v/v) H2SO4 , followed by heating at 1208 for The PhytoLibraryTM kit was purchased from PURIMED Co (Seoul, Korea) Optical rotations: Jasco P-1010 polarimeter at 258 1D- and 2DNMR experiments: Bruker Avance-400 FT-NMR instrument; with Me4Si as internal standard HR-EIMS and ESI-MS: Jeol JMS-700-M mass spectrometer and an API 3200-Q-TRAP LC/MS/MS system, resp Plant Material The roots of Asarum sieboldii Miquel (Aristolochiaceae) were purchased from Sun Ten Pharmaceutical Co., Ltd (Taipei, Taiwan) A voucher specimen (D10) was deposited at the Herbarium of College of Oriental Medicine, Kyung Hee University, Korea Extraction and Isolation The roots of A sieboldii (3 kg) were extracted with MeOH (5 10 l) for 24 h by percolation The solvent was evaporated in vacuo to afford a MeOH extract (300 g), which was suspended in H2O (1 l), and extracted successively with hexane (5 l), AcOEt (5 l), and BuOH (3 l) Compound (2.3 g, 0.077%) [14] was isolated from the hexane layer by precipitation The soluble hexane extract (100 g) was fractionated by FC (1 kg SiO2 ; hexane/CHCl3 10 : 0, 19 : 1, : 1, : 1, : 1, then neat MeOH, l each) to afford 15 fractions: Fr I – Fr XV Safrole (3.7 g, 0.12%) [29], methyleugenol (14.3 g, 0.47% w/w) [12], and (1.1 g, 0.036%) [14] were also isolated from the first separation Fr VII (6 g), eluted with hexane/CHCl3 : 1, was subjected to FC (150 g SiO2 ; hexane/AcOEt 99 : 1, 98 : 2, 95 : 5, then neat MeOH, l each) to provide asaricin (459 mg, 0.015%) [30] Fr VIII (15 g) was purified by FC (300 g SiO2 ; hexane/AcOEt 99 : 1, 98 : 2, 95 : 5, then neat MeOH, l each) to afford 13,5-dimethoxytoluene3 (189 mg, 0.0063%) [28] and (59 mg, 0.0019%) [10] Fr XI and Fr XII (12 g), eluted with hexane/CHCl3 : 1, were further fractionated by FC (300 g SiO2 ; hexane/AcOEt 99 : 1, 98 : 2, 95 : 5, : : 1, then neat MeOH, l each) to yield (154 mg, 0.0051%) [26] and (389 mg, 0.013%) [27] Fr XIII (17 g), eluted with hexane/CHCl3 : 1, was further separated by FC (400 g SiO2 ; linear gradient of 0, 0.1, and 0.2% MeOH in CHCl3 , then neat MeOH, l each) to afford (1.0 g, 0.033%) [14] Measurement of NO Production in LPS-Activated BV-2 Microglial Cells Measurement of NO formation by iNOS was performed in cultured BV-2 microglial cells The cells were maintained in Dulbecco3s Modified Eagle Medium (DMEM) supplemented with penicillin-streptomycin and 10% fetal bovine serum (FBS) at 378 in humidified air containing 5% of CO2 To evaluate the inhibitory activity of the test materials on LPS-induced NO production, the cells in 10% FBS-DMEM, without Phenol Red, 350 CHEMISTRY & BIODIVERSITY – Vol (2008) were plated in 24-well plates (5 105 cells/ml), and then incubated for 24 h After incubation, the cells were washed with PBS, replaced with new medium, and then incubated in the medium with mg/ml of LPS in the presence or absence of test sample After an additional 20-h incubation, the media were collected and analyzed for nitrite accumulation as an indicator of NO production by the Griess reaction Briefly, 100 ml of Griess reagent (0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in H2O and 1% sulfanilamide in 5% H3PO4 ) were added to 100 ml of each supernatant from LPS-treated or LPS-andsample-treated cells in 96-well plates The UV/VIS absorbance was measured at 540 nm using a microplate 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Chen, T Fu, T Tao, J Yang, Y Chang, M Wang, L Kim, L Qu, J Cassady, R Scalzo, X Wang, J Nat Prod 2005, 68, 773 Received June 7, 2007 ... 75.9% inhibition at the maximum test concentration of mg/ml (5.49 10 m) Figure Effects of compounds – on LPS-induced NO production in BV-2 microglial cells Inhibition of NO production was calculated... IC50 Values, the sample concentration resulting in 50% inhibition of NO production, were determined by non-linear regression analysis (% inhibition vs concentration) MTT Assay for Cell Viability... supplemented with penicillin-streptomycin and 10% fetal bovine serum (FBS) at 378 in humidified air containing 5% of CO2 To evaluate the inhibitory activity of the test materials on LPS-induced NO production,